Experimental Study On Effect Of DMSO Upon Antimicrobial Lethality And Associated Mechanism

Objectives To reveal the overall effect of DMSO on bactericidal antimicrobial lethality, and to explore the underlying mechanism, aiming to set a cutoff for DMSO of dissolving antibiotics and to provide experimental guidelines for fair evaluation of antibiotic efficacy.Methods Assays of growth curve determination, minimal inhibitory concentration(MIC) determination, time-kill or concentration-kill curve(bar) were recruited to exhibit effect of DMSO on antimicrobial lethality tobugs. Meanwhile, antimicrobial lethality in co-administration with DMSO was treated on clinical isolates of E. coli ATCC25922 and A.Baunannii ATCC17978. Then Δkat G Δkat E double mutant constructed by sequential marker excision and bacteriophage P1 was treated with DMSO and antimicrobials to test whether genes encode hydroperixidase I and II participate in the protection by DMSO on antibiotic killing.E. coli cells were labeled by carboxy-H2 DCFDA, a fluorescent dye that reacts with all 3 ROS and generates high and stable fluorescent labeling intracellularly. Then intracellular reactive oxygen species(ROS) were detected by both flow cytometry and fluorescent microscope. To add, assay to determine whether and how much DMSO got involved in perturbing ROS-mediated programmed cell death was conducted to co-demonstrate the underlying mechanism.Results 1. DMSO concentrations of 3%, 5% and 7.5% affected little on bacteria growth especially on exponential growth. 2.In the presence of DMSO at indicated concentrations, killing actions of all three classes of antibiotics(oxolinic acid, ciprofloxacin, ampicillin and kanamycin) were partially blocked or delayed by DMSO. Similarly, DMSO affected some antimicrobial efficacy in treating E.coli ATCC25922 and A. baumannii ATCC17978.3. The protective effect of DMSO could also be witnessed when Δkat GΔkat E double deficiency mutant cells were treated with oxolinic acid, ampicillin and kanamycin. However, DMSO turned to accelerate but not to hinder ciprofloxacin killing process. 4. In flow cytometry assay, oxolinic acid-triggered surges in intracellular ROS accumulation was partially eliminated by DMSO, and similar results were obtained in fluorescent microscope detection. 5. DMSO interfered not only antibiotic liquid killing but also post-stress ROS-mediated PCD.Conclusions 1. DMSO improved bacteria survival against bactericidal drugs not simply due to inhibition on bacteria growth which may lead to phenotypic tolerance to antimicrobial treatment. 2. DMSO protection was shown to be independent of both type I and type II hydroperixidase. 3. DMSO protected E. coli cells from bactericidal antimicrobial treatment mainly by scavenging intracellular ROS, which resulted in a decrease of detrimental hydroxyl radicals, perturbation of PCD and a survival improvement.